Culture effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on cryopreserved human adipose-derived stromal/stem cell proliferation and adipogenesis

Abstract

Previous studies have demonstrated that EGF and bFGF maintain the stem cell properties of proliferating human adipose-derived stromal/stem cells (hASCs) in vitro. While the expansion and cryogenic preservation of isolated hASCs are routine, these manipulations can impact their proliferative and differentiation potential. This study examined cryogenically preserved hASCs (n = 4 donors), with respect to these functions, after culture with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) at varying concentrations (0-10 ng/ml). Relative to the control, cells supplemented with EGF and bFGF significantly increased proliferation by up to three-fold over 7-8 days. Furthermore, cryopreserved hASCs expanded in the presence of EGF and bFGF displayed increased oil red O staining following adipogenic induction. This was accompanied by significantly increased levels of several adipogenesis-related mRNAs: aP2, C/EBPalpha, lipoprotein lipase (LPL), PPARgamma and PPARgamma co-activator-1 (PGC1). Adipocytes derived from EGF- and bFGF-cultured hASCs exhibited more robust functionality based on insulin-stimulated glucose uptake and atrial natriuretic peptide (ANP)-stimulated lipolysis. These findings indicate that bFGF and EGF can be used as culture supplements to optimize the proliferative capacity of cryopreserved human ASCs and their adipogenic differentiation potential.

Figure 1

Effects of growth factors on adipogenesis. Photomicrographs of cells from one representative donor grown in 16 different EGF- and bFGF-supplemented conditions (EGF concentrations on x axis, bFGF concentrations on y axis) prior to induction of adipogenesis. Similar photomicrographs taken for three other donors are not shown. The cells are stained with oil red O. Magnification, ×100

Figure 2

Effects of growth factors on oil red O staining. Human ASCs from n = 3 donors were preconditioned for 8 days in the absence or presence of EGF and bFGF at concentrations of 0.1, 1.0 or 10 ng/ml of both reagents. The hASCs under the different conditions were then induced to undergo adipogenesis. Following adipogenic differentiation for 9 days, the hASCs were stained with oil red O. The retained dye was eluted from the adherent cells and quantified by absorbance reading at OD₅₁₀. Unfixed and unstained representative wells maintained under each of the EGF/bFGF preconditions and under the subsequent adipogenic culture condition were trypsinized and the total number of cells per well determined. The mean ± SD (n = 3 donors) oil red O absorbance was normalized relative to 10⁶ hASCs, as indicated on the y axis. The OD₅₁₀ values are displayed relative to the preconditioning EGF/bFGF concentrations (x axis)

Figure 3

Effects of growth factors on adipogenic mRNA levels. Results of quantitative PCR analysis for cells grown in EGF- and bFGF-supplemented conditions prior to the induction of adipogenesis. Quantities were normalized to cyclophilin B. Values are the mean ± SD fold increase for n = 3 donors. (A) Values for PPARγ2 mRNA from hASCs preconditioned with the indicated levels of EGF/bFGF in the absence (left panel) and presence (right panel) of adipogenic induction. All levels are expressed as fold increase relative to the level of hASCs preconditioned in the absence of EGF/bFGF and induced for adipogenesis; this value is defined as ‘1’ relative inductive unit (RIU) on the y axis. (B) Relative mRNA values for the adipogenic biomarkers aP2 (fatty acid binding protein 4), C/EBPα (CAAT/enhancer binding protein α), LPL (lipoprotein lipase), and PGC1 (PPARγ co-activator 1) in hASCs induced to undergo adipogenesis. All mRNA values for these same biomarkers in hASCs without adipogenic induction were two or three orders of magnitude lower (data not shown). Values on the x axis refer to concentrations of EGF/bFGF in ng/ml. Statistical significance relative to the hASC preconditioned in the absence of EGF and bFGF and induced to undergo adipogenesis is indicated by ^*(p < 0.05)

Figure 4

Effects of EGF and FGF on lipolysis. Lipolytic response of adipocytes preconditioned prior to adipogenesis in the absence (top panel) or presence (bottom panel) of 10 ng/ml EGF and 10 ng/ml FGF were compared in response to increasing concentrations of ANP (right panels) and isoproterenol (left panels). Average lipolysis (with maximum and minimum values) for n = 2 donors is shown